Ca2+?activated potassium channels, such as small? and intermediate?conductance K+ channels (SK and IK), are widely expressed in excitable tissues. They play pivotal roles in regulating membrane excitability by Ca2+. Unlike voltage?gated K+ channels, activation of SK/IK channels is achieved exclusively by Ca2+. Calmodulin (CaM), tethered to the channel C?terminus, serves as the high?affinity Ca2+ sensor. Four EF?hands, two located at the CaM N?terminus (N?lobe) and the other two at the C?terminus (C?lobe), are the high affinity Ca2+ binding sites. The Ca2+?mediated interaction between CaM and the CaM binding domain (CaMBD) activates the channel. In addition to their physiological roles, SK/IK channels have been implicated in clinical abnormalities. Consequently, a tremendous effort has been devoted to developing small molecules targeting SK/IK channels in both academia and industry. 1?ethyl?2?benzimidazolinone (1?EBIO) is such a prototype that potentiates the SK/IK channel activity and effectively decreases the membrane excitability. Studies have shown that the 1? EBIO compounds are beneficial in disease models of the central nervous system and the cardiovascular system. In general, however, the 1?EBIO compounds suffer from drawbacks, such as lack of selectivity, which hamper their potential for clinical trials. A key contributing factor is lack of knowledge of how the 1?EBIO compounds interact with their binding site and achieve their effects on SK/IK channels. Until the publication of our recent papers, it was not known where these compounds might interact with SK/IK channels. The molecular properties of the drug binding site remain unclear and it is not known how the 1?EBIO compounds work, or how these compounds achieve their selectivity. To uncover the molecular properties of the drug binding site for these compounds and understand the molecular mechanisms for the actions of the 1?EBIO compounds, we will use integrated approaches of structural biology, molecular biology, biochemistry biophysics and electrophysiology. Specifically, we will focus on the following issues: (1) Characterization of the binding site for the 1?EBIO compounds and molecular mechanisms that contribute to selectivity of the 1?EBIO compounds for SK/IK channels. 1?EBIO compounds over different types of SK/IK channels. (2) Molecular mechanisms by which the 1?EBIO compounds potentiate SK/IK channels. (3) Molecular mechanisms by which the 1?EBIO compounds inhibit SK/IK channels. Results from the proposed work will provide the molecular properties of the drug binding site for the 1?EBIO compounds. The results will fill in the current knowledge gap regarding how these compounds modulate SK/IK channel activity (potentiation and inhibition). The knowledge will facilitate development of future generations of therapeutics targeting SK/IK channels by structure?based drug design/development. Broadly, our results will help develop compounds targeting other CaM?target protein complexes involved in Ca2+ dependent signaling as well as new drugs targeting membrane lipids beyond the ion channel field.